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Amplification simply means “Multiplying” - DNA amplification means “Multiplying DNA multifolds”
Amplification Technique - Classification
By Thermal Methods
1. Thermal Cycling
- PCR
- LCR
2. Isothermal Cycling
- NASBA
- B-DNA
By Multiplication Folds
1. Target Amplification
- PCR
- NASBA
2. Probe Amplification
LCR
- Q β
- Replicase
3. Signal Amplification
B-DNA
Polymerase Chain Reaction
→ Revolutionary technique invented by Karry B. Mullis - Noble Prize in 1993.
→ The polymerase chain reaction -PCR is a Test Tube Method for amplifying a selected DNA sequence.
→ End-Product by amplification referred as Amplicon.
Technique Features
→ Target Amplification by Thermal cycling.
→ Amplification is Expontential - No. of samples after ‘n’ number of cycles = 2n
→ Ex. 20 cycles result in Million-fold Amplification of Target DNA.
→ Duration: One cycle of PCR require 5 to 10 mins.
Pre-Requisites for PCR
→ Sample DNA to be amplified.
→ Deoxynucleotides.
→ Thermostable Polymerase - Taq Polymerase obtained from Thermus Aquaticus found in hot springs.
→ Primer → For quantitative RNA analyses, after RNA copying & mRNA quantitation (By RT-PCR method).
→ To score in vivo protein - DNA occupancy using “chromatin immuno-precipitation assays (CHIP)”.
→ To facilitate New Generation Sequencing, by amplifying the DNA.
→ MgCl2 & KCI
Variations of PCR
Reverse Transcriptase PCR (RT PCR)
→ PCR amplification of a Reverse Transcription Product.
→ Amplifies very small amounts of RNA (mRNA, rRNA, tRNA etc.,)
→ Uses: Useful for obtaining Relative Gene Expression in a cell.
Real Time / Homogenous / Kinetic PCR
→ Type of Quantitative PCR.
→ Simultaneously Amplifies & Quantitate the amount of Target Sequence in REAL TIME PCR itself.
→ Real time: Refers to “During exponential phase of PCR cycle & not after amplification is over”.
Nested PCR
→ Used if, DNA to be amplified is present in Low Concentration, compared to Total DNA in Sample.
→ Uses 2 sets of Amplification Primers, for amplification of Single Target, in 2 PCR runs.
Multiplex PCR
→ Simultaneous Amplification of Multiple Targets in Single Reaction.
→ Uses more than 1 pair of Primer.
Arbitrary PCR / Arbitrarily Primed (AP-PCR)
“Randomly amplified Polymorphic DNA / Random Amplification of Polymorphic DNA (RAPD)”
→ Uses Short Random Primers (Usually 10-15 Bases)
→ Amplifies Anonymous DNA Stretches under Low Stringency.
→ Low Reproducibility.
Application of PCR
→ Used in Forensic Medicine.
→ Offers Rapid & Sensitive method for Detecting Viral RNA / DNA Sequences .
→ Real time PCR determines Viral Load.
→ Prenatal Diagnosis (Ex. Cystic Fibrosis).
→ Detecting Length Polymorphisms (Ex. VNTR).
→ Allows sufficient mutant DNA synthesis to be sequenced for Mutation Detections.
→ Establish precise tissue types for transplants.
→ Study evolution, using DNA from archeological samples.